Lack of neutralization of Chlamydia trachomatis infection by high avidity monoclonal antibodies to surface-exposed major outer membrane protein variable domain IV.

Chlamydia trachomatis is the leading cause of sexually transmitted diseases causing frequent, long-lasting, and often asymptomatic recurrent infections resulting in severe reproductive complications. C. trachomatis is an intracellular Gram-negative bacterium with a biphasic developmental cycle in which extracellular, infectious elementary bodies (EB) alternate with the intracellular replicating reticulate bodies (RB). The outer membrane of EB consists of a tight disulfide cross-linking protein network. The most essential protein is the 42 kDa major outer membrane protein (MOMP) that contributes to the rigid structural integrity of the outer membrane. MOMP is a transmembrane protein with a ß-barrel structure consisting of four variable domains (VD) separated by five constant domains. VDIV possesses surface-exposed species-specific epitopes recognized by the immune system and, therefore, functions as a candidate for vaccine development. To analyze the protective contribution of antibodies for a MOMP vaccine, we investigated the specificity and binding characteristics of two monoclonal antibodies (MAb)224.2 and MAb244.4 directed against C. trachomatis serovar D MOMP. By immunoelectron microscopy, we found that both MAb bind to the surface of C. trachomatis EB. By epitope mapping, we characterized the MOMP epitope as linear consisting of 6 amino acids: 322TIAGAGD328. By ELISA it was shown that both antibodies bind with a higher avidity to the chlamydial surface compared to binding to monomeric MOMP, indicating that the antibodies bind divalently to the surface of C. trachomatis EB. Despite strong binding to the chlamydial surface, the antibodies only partially reduced the infectivity. This may be explained by the observation that even though both MAb covered the EB surface, antibodies could not be regularly detected on EB after the uptake into the host cell.

Detection of large extracellular silver nanoparticle rings observed during mitosis using darkfield microscopy.

During studies on the absorption and interactions between silver nanoparticles and mammalian cells grown in vitro it was observed that large extracellular rings of silver nanoparticles were deposited on the microscope slide, many located near post-mitotic cells. Silver nanoparticles (AgNP, 80nm), coated with citrate, were incubated at concentrations of 0.3 to 30 µg/ml with a human-derived culture of retinal pigment epithelial cells (ARPE-19) and observed using darkfield and fluorescent microscopy, 24 h after treatment. Approximately cell-sized extracellular rings of deposited AgNP were observed on the slides among a field of dispersed individual AgNP. The mean diameter of 45 nanoparticles circles was 62.5 +/-12 microns. Ring structures were frequently observed near what appeared to be post-mitotic daughter cells, giving rise to the possibility that cell membrane fragments were deposited on the slide during mitosis, and those fragments selectively attracted and retained silver nanoparticles from suspension in the cell culture medium. These circular structures were observable for the following technical reasons: 1) darkfield microscope could observe single nanoparticles below 100 nm in size, 2) a large concentration (108 and 109) of nanoparticles was used in these experiments 3) negatively charged nanoparticles were attracted to adhesion membrane proteins remaining on the slide from mitosis. The observation of silver nanoparticles attracted to apparent remnants of cellular mitosis could be a useful tool for the study of normal and abnormal mitosis.

Biophysical comparison of four silver nanoparticles coatings using microscopy, hyperspectral imaging and flow cytometry.

This study compared the relative cellular uptake of 80 nm silver nanoparticles (AgNP) with four different coatings including: branched polyethyleneimine (bPEI), citrate (CIT), polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG). A gold nanoparticle PVP was also compared to the silver nanoparticles. Biophysical parameters of cellular uptake and effects included flow cytometry side scatter (SSC) intensity, nuclear light scatter, cell cycle distributions, surface plasmonic resonance (SPR), fluorescence microscopy of mitochondrial gross structure, and darkfield hyperspectral imaging. The AgNP-bPEI were positively charged and entered cells at a higher rate than the negatively or neutrally charged particles. The AgNP-bPEI were toxic to the cells at lower doses than the other coatings which resulted in mitochondria being transformed from a normal string-like appearance to small round beaded structures. Hyperspectral imaging showed that AgNP-bPEI and AgNP-CIT agglomerated in the cells and on the slides, which was evident by longer spectral wavelengths of scattered light compared to AgNP-PEG and AgNP-PVP particles. In unfixed cells, AgNP-CIT and AgNP-bPEI had higher SPR than either AgNP-PEG or AgNP-PVP particles, presumably due to greater intracellular agglomeration. After 24 hr. incubation with AgNP-bPEI, there was a dose-dependent decrease in the G1 phase and an increase in the G2/M and S phases of the cell cycle suggestive of cell cycle inhibition. The nuclei of all the AgNP treated cells showed a dose-dependent increase in nanoparticles following non-ionic detergent treatment in which the nuclei retained extra-nuclear AgNP, suggesting that nanoparticles were attached to the nuclei or cytoplasm and not removed by detergent lysis. In summary, positively charged AgNP-bPEI increased particle cellular uptake. Particles agglomerated in the peri-nuclear region, increased mitochondrial toxicity, disturbed the cell cycle, and caused abnormal adherence of extranuclear material to the nucleus after detergent lysis of cells. These results illustrate the importance of nanoparticle surface coatings and charge in determining potentially toxic cellular interactions.

Determination of Silver Nanoparticle Dose in vitro.

An important issue for interpreting in vitro nanomaterial testing is quantifying the dose delivered to target cells. Considerations include the concentration added to the culture, the proportion of the applied dose that interacts with the target cells, and the amount that is eventually absorbed by the target cells. Rapid and efficient techniques are needed to determine delivered doses. Previously, we demonstrated that TiO2 and silver nanoparticles (AgNP) were absorbed by cells in a dose dependent manner between 1 µg/ml and 30 µg/ml and were detected in cells by light scatter using a flow cytometer. Here, we compare four potential indices of the dose of AgNP to cells, including: inductively coupled plasma - mass spectrometry (ICP-MS); flow cytometry side scatter (SSC); and amount of silver deposited to the cell layer as estimated with both an integrated Volumetric Centrifugation Method - In Vitro Sedimentation, Diffusion and Dosimetry Model (VCM-ISDD) and a Distorted Grid (DG) model. A retinal pigment epithelial cell line was exposed to 20 nm or 75 nm citrate-coated AgNP for 24 hr. The relationships between particle sizes and internalized doses varied according to the dose metric. Twenty-four hours after exposure, the cell layer contained a greater mass of silver when treated with 75 nm AgNP than with 20 nm AgNP. When the dose was expressed as the number of particles or as the total surface area of absorbed particles, however, the reverse was true; the dose to the cells was higher after exposure to 20 than 75 nm AgNP. Flow cytometry SSC increased with dose for both sizes of AgNP, and was correlated with Ag in cells measured by ICP-MS. The rate of SSC increase was greater for 75 than for 20 nm AgNP, suggesting it could be used as an indicator of cellular dose after accounting for particle size and composition. Silver was detected by ICP-MS in re-suspended supernates of the isolated cell layer suggested that not all the silver deposited to the cell layer was absorbed by the cells. Both the VCM-ISDD and DG models estimated the proportion of Ag deposited to the cellular layer, which in both cases was greater than the amount of silver in the cells measured by ICP-MS. Modeled deposition more closely compared to the total Ag deposition by ICP-MS, i.e. mass of silver in the cells plus the resuspended, unabsorbed Ag from the cell layer. ICP-MS indicated the mass of silver in cells from AgNP treatment, but not whether the Ag was in the form of particles or dissolved ions. Deposition models predicted the amount of AgNP deposited to the cell layer, but not cellular uptake. Flow cytometry SSC was correlated to cellular uptake of particle-form AgNP and could be calibrated against ICP-MS to indicate mass of cellular uptake. Therefore, a combination of approaches may be required to accurately understand cellular dosimetry of in vitro nanotoxicology experiments. In summary, cellular dosimetry is an important consideration for nanotoxicology experiments, and not necessarily related to the applied dose.

Moderate perinatal thyroid hormone insufficiency alters visual system function in adult rats.

Thyroid hormone (TH) is critical for many aspects of neurodevelopment and can be disrupted by a variety of environmental contaminants. Sensory systems, including audition and vision are vulnerable to TH insufficiencies, but little data are available on visual system development at less than severe levels of TH deprivation. The goal of the current experiments was to explore dose-response relations between graded levels of TH insufficiency during development and the visual function of adult offspring. Pregnant Long Evans rats received 0 or 3 ppm (Experiment 1), or 0, 1, 2, or 3 ppm (Experiment 2) of propylthiouracil (PTU), an inhibitor of thyroid hormone synthesis, in drinking water from gestation day (GD) 6 to postnatal day (PN) 21. Treatment with PTU caused dose-related reductions of serum T4, with recovery on termination of exposure, and euthyroidism by the time of visual function testing. Tests of retinal (electroretinograms; ERGs) and visual cortex (visual evoked potentials; VEPs) function were assessed in adult offspring. Dark-adapted ERG a-waves, reflecting rod photoreceptors, were increased in amplitude by PTU. Light-adapted green flicker ERGs, reflecting M-cone photoreceptors, were reduced by PTU exposure. UV-flicker ERGs, reflecting S-cones, were not altered. Pattern-elicited VEPs were significantly reduced by 2 and 3 ppm PTU across a range of stimulus contrast values. The slope of VEP amplitude-log contrast functions was reduced by PTU, suggesting impaired visual contrast gain. Visual contrast gain primarily reflects function of visual cortex, and is responsible for adjusting sensitivity of perceptual mechanisms in response to changing visual scenes. The results indicate that moderate levels of pre-and post-natal TH insufficiency led to alterations in visual function of adult rats, including both retinal and visual cortex sites of dysfunction.

CD11c-targeted Delivery of DNA to Dendritic Cells Leads to cGAS- and STING-dependent Maturation.

Immunotherapeutic activation of tumor-specific T cells has proven to be an interesting approach in anticancer treatment. Particularly, anti-CTLA-4 and anti-PD-1/PD-L1 treatment looks promising, and conceivably, even better clinical results might be obtained if such treatment could be combined with boosting the existing tumor-specific T-cell response. One way to achieve this could be by increasing the level of maturation of dendritic cells locally and in the draining lymph nodes. When exposed to cancer cells, dendritic cells may spontaneously mature because of danger-associated molecular patterns derived from the tumor cells. Double-stranded DNA play a particularly important role in the activation of the dendritic cells, through engagement of intracellular DNA-sensors, and signaling through the adaptor protein STING. In the present study, we have investigated the maturational response of human monocyte-derived dendritic cells (moDC) and human monocytic THP-1 cells to targeted and untargeted DNA. We used an anti-CD11c antibody conjugated with double-stranded DNA to analyze the maturation status of human moDCs, as well as maturation using a cGAS KO and STING KO THP-1 cell maturation model. We found that dendritic cells can mature after exposure to cytoplasmic double-stranded DNA delivered through CD11c-mediated endocytosis. Moreover, we show that THP-1 cells matured using IL-4, GM-CSF, and ionomycin upregulate DC-maturation markers after CD11c-targeted delivery of double-stranded DNA. This upregulation is completely abrogated in cGAS KO and STING KO cells.

Toluene inhalation exposure for 13 weeks causes persistent changes in electroretinograms of Long-Evans rats.

Studies of humans chronically exposed to volatile organic solvents have reported impaired visual functions, including low contrast sensitivity and reduced color discrimination. These reports, however, lacked confirmation from controlled laboratory experiments. To address this question experimentally, we examined visual function by recording visual evoked potentials (VEP) and/or electroretinograms (ERG) from four sets of rats exposed repeatedly to toluene. In addition, eyes of the rats were examined with an ophthalmoscope and some of the retinal tissues were evaluated for rod and M-cone photoreceptor immunohistochemistry. The first study examined rats following exposure to 0, 10, 100 or 1000ppm toluene by inhalation (6hr/d, 5d/wk) for 13 weeks. One week after the termination of exposure, the rats were implanted with chronically indwelling electrodes and the following week pattern-elicited VEPs were recorded. VEP amplitudes were not significantly changed by toluene exposure. Four to five weeks after completion of exposure, rats were dark-adapted overnight, anesthetized, and several sets of electroretinograms (ERG) were recorded. In dark-adapted ERGs recorded over a 5-log (cd-s/m(2)) range of flash luminance, b-wave amplitudes were significantly reduced at high stimulus luminance values in rats previously exposed to 1000ppm toluene. A second set of rats, exposed concurrently with the first set, was tested approximately one year after the termination of 13 weeks of exposure to toluene. Again, dark-adapted ERG b-wave amplitudes were reduced at high stimulus luminance values in rats previously exposed to 1000ppm toluene. A third set of rats was exposed to the same concentrations of toluene for only 4 weeks, and a fourth set of rats exposed to 0 or 1000ppm toluene for 4 weeks were tested approximately 1year after the completion of exposure. No statistically significant reductions of ERG b-wave amplitude were observed in either set of rats exposed for 4 weeks. No significant changes were observed in ERG a-wave amplitude or latency, b-wave latency, UV- or green-flicker ERGs, or in photopic flash ERGs. There were no changes in the density of rod or M-cone photoreceptors. The ERG b-wave reflects the firing patterns of on-bipolar cells. The reductions of b-wave amplitude after 13 weeks of exposure and persisting for 1year suggest that alterations may have occurred in the inner nuclear layer of the retina, where the bipolar cells reside, or the outer or inner plexiform layers where the bipolar cells make synaptic connections. These data provide experimental evidence that repeated exposure to toluene may lead to subtle persistent changes in visual function. The fact that toluene affected ERGs, but not VEPs, suggests that elements in the rat retina may be more sensitive to organic solvent exposure than the rat visual cortex.

Neurophysiological assessment of auditory, peripheral nerve, somatosensory, and visual system function after developmental exposure to gasoline, E15, and E85 vapors.

The use of gasolines blended with a range of ethanol concentrations may result in inhalation of vapors containing a variable combination of ethanol with other volatile gasoline constituents. The possibility of exposure and potential interactions between vapor constituents suggests the need to evaluate the possible risks of this complex mixture. Previously we evaluated the effects of developmental exposure to ethanol vapors on neurophysiological measures of sensory function as a component of a larger project evaluating developmental ethanol toxicity. Here we report an evaluation using the same battery of sensory function testing in offspring of pregnant dams exposed during gestation to condensed vapors of gasoline (E0), gasoline blended with 15% ethanol (E15) or gasoline blended with 85% ethanol (E85). Pregnant Long-Evans rats were exposed to target concentrations 0, 3000, 6000, or 9000 ppm total hydrocarbon vapors for 6.5h/day over GD9 - GD20. Sensory evaluations of male offspring began as adults. The electrophysiological testing battery included tests of: peripheral nerve (compound action potentials, nerve conduction velocity [NCV]), somatosensory (cortical and cerebellar evoked potentials), auditory (brainstem auditory evoked responses), and visual functions. Visual function assessment included pattern elicited visual evoked potentials (VEP), VEP contrast sensitivity, dark-adapted (scotopic) electroretinograms (ERGs), light-adapted (photopic) ERGs, and green flicker ERGs. The results included sporadic statistically significant effects, but the observations were not consistently concentration-related and appeared to be statistical Type 1 errors related to multiple dependent measures evaluated. The exposure concentrations were much higher than can be reasonably expected from typical exposures to the general population during refueling or other common exposure situations. Overall the results indicate that gestational exposure of male rats to ethanol/gasoline vapor combinations did not cause detectable changes in peripheral nerve, somatosensory, auditory, or visual function when the offspring were assessed as adults.

Use of novel inhalation kinetic studies to refine physiologically-based pharmacokinetic models for ethanol in non-pregnant and pregnant rats.

Ethanol (EtOH) exposure induces a variety of concentration-dependent neurological and developmental effects in the rat. Physiologically-based pharmacokinetic (PBPK) models have been used to predict the inhalation exposure concentrations necessary to produce blood EtOH concentrations (BEC) in the range associated with these effects. Previous laboratory reports often lacked sufficient detail to adequately simulate reported exposure scenarios associated with BECs in this range, or lacked data on the time-course of EtOH in target tissues (e.g. brain, liver, eye, fetus). To address these data gaps, inhalation studies were performed at 5000, 10 000, and 21 000 ppm (6 h/d) in non-pregnant female Long-Evans (LE) rats and at 21 000 ppm (6.33 h/d) for 12 d of gestation in pregnant LE rats to evaluate our previously published PBPK models at toxicologically-relevant blood and tissue concentrations. Additionally, nose-only and whole-body plethysmography studies were conducted to refine model descriptions of respiration and uptake within the respiratory tract. The resulting time-course and plethysmography data from these in vivo studies were compared to simulations from our previously published models, after which the models were recalibrated to improve descriptions of tissue dosimetry by accounting for dose-dependencies in pharmacokinetic behavior. Simulations using the recalibrated models reproduced these data from non-pregnant, pregnant, and fetal rats to within a factor of 2 or better across datasets, resulting in a suite of model structures suitable for simulation of a broad range of EtOH exposure scenarios.

Neurophysiological assessment of auditory, peripheral nerve, somatosensory, and visual system functions after developmental exposure to ethanol vapors.

Ethanol-blended gasoline entered the market in response to demand for domestic renewable energy sources, and may result in increased inhalation of ethanol vapors in combination with other volatile gasoline constituents. It is important to understand potential risks of inhalation of ethanol vapors by themselves, and also as a baseline for evaluating the risks of ethanol combined with a complex mixture of hydrocarbon vapors. Because sensory dysfunction has been reported after developmental exposure to ethanol, we evaluated the effects of developmental exposure to ethanol vapors on neurophysiological measures of sensory function as a component of a larger project evaluating developmental ethanol toxicity. Pregnant Long-Evans rats were exposed to target concentrations 0, 5000, 10,000, or 21,000 ppm ethanol vapors for 6.5h/day over GD9-GD20. Sensory evaluations of male offspring began between PND106 and PND128. Peripheral nerve function (compound action potentials, nerve conduction velocity (NCV)), somatosensory (cortical and cerebellar evoked potentials), auditory (brainstem auditory evoked responses), and visual evoked responses were assessed. Visual function assessment included pattern elicited visual evoked potentials (VEPs), VEP contrast sensitivity, and electroretinograms recorded from dark-adapted (scotopic), light-adapted (photopic) flashes, and UV flicker and green flicker. No consistent concentration-related changes were observed for any of the physiological measures. The results show that gestational exposure to ethanol vapor did not result in detectable changes in peripheral nerve, somatosensory, auditory, or visual function when the offspring were assessed as adults.

In vitro phototoxicity and hazard identification of nano-scale titanium dioxide.

Titanium dioxide nanoparticles (nano-TiO(2)) catalyze reactions under UV radiation and are hypothesized to cause phototoxicity. A human-derived line of retinal pigment epithelial cells (ARPE-19) was treated with six samples of nano-TiO(2) and exposed to UVA radiation. The TiO(2) nanoparticles were independently characterized to have mean primary particle sizes and crystal structures of 22nm anatase/rutile, 25nm anatase, 31nm anatase/rutile, 59nm anatase/rutile, 142nm anatase, and 214nm rutile. Particles were suspended in cell culture media, sonicated, and assessed for stability and aggregation by dynamic light scattering. Cells were treated with 0, 0.3, 1, 3, 10, 30, or 100µg/ml nano-TiO(2) in media for 24hrs and then exposed to UVA (2hrs, 7.53J/cm(2)) or kept in the dark. Viability was assessed 24hrs after the end of UVA exposure by microscopy with a live/dead assay (calcein-AM/propidium iodide). Exposure to higher concentrations of nano-TiO(2) with UVA lowered cell viability. The 25nm anatase and 31nm anatase/rutile were the most phototoxic (LC(50) with UVA<5µg/ml), while the 142nm anatase and 214nm rutile were the least phototoxic. An acellular assay ranked TiO(2) nanoparticles for their UVA photocatalytic reactivities. The particles were found to be capable of generating thiobarbituric acid reactive substances (TBARS) under UVA. Flow cytometry showed that nano-TiO(2) combined with UVA decreased cell viability and increased the generation of reactive oxygen species (ROS, measured by Mitosox). LC(50) values under UVA were correlated with TBARS reactivity, particle size, and surface area.

Acute inhalation of 2,2,4-trimethylpentane alters visual evoked potentials and signal detection behavior in rats.

The volatile organic compound 2,2,4-trimethylpentane (TMP, "isooctane") is a constituent of gasoline for which the current health effects data are insufficient to permit the US Environmental Protection Agency to conduct a risk assessment. The potential neurological impairment from acute inhalation exposure to TMP was evaluated in adult male Long-Evans rats using both electrophysiological and behavioral assessments. Visual evoked potentials (VEPs) were recorded from rats viewing modulated visual patterns (0.16 cycles per degree visual angle (cpd), 60% contrast, 4.55Hz appear/disappear). Rats (n=7-10/dose) were exposed to TMP vapors in concentrations of 0, 500, or 1000 ppm for 60-min. A VEP was recorded before exposure and at 10 min intervals during exposure and also for 60 min after exposure terminated. The spectral amplitude of the frequency-double component (F2) was significantly reduced after exposure to TMP. In behavioral assessments, rats (n=14) performed an appetitively motivated visual signal detection task while breathing 0, 500, 1500, 1000, 2000, or 2500 ppm TMP for 62 min. Slight reductions in accuracy of performance were observed at the 2500 ppm concentration. Concentrations of TMP in the brain were estimated using a physiologically based pharmacokinetic (PBPK) model to be less than 0.2mM after 62 min at 2500 ppm. Together these data demonstrate that TMP, like other volatile organic substances, impairs neurological function during acute inhalation exposure and that the small magnitude of the observed effects is consistent with the low concentrations of this hydrocarbon that were estimated to reach the CNS.